The strength of synaptic transmission is a critical determinant of information processing in neural circuits. Evoked neurotransmission depends on localized Ca2+ influx triggering neurotransmitter release from synaptic vesicles at specialized domains of presynaptic terminals called active zones. At the active zone membrane, synaptic vesicles are docked and molecularly primed to respond to a rise in Ca2+ concentration by fusing with the membrane to release neurotransmitter. A conserved complex of active zone-associated proteins makes up the active zone cytomatrix. In Drosophila melanogaster, these proteins include the ELKS family protein Bruchpilot, Rab3-interacting molecule (RIM), RIM-binding protein, Unc13, and Fife. Active zone cytomatrix proteins contain many lipid- and protein-binding domains that mediate diverse interactions with key players in synaptic transmission, leading to the model that the active zone cytomatrix spatially organizes presynaptic terminals for the millisecond coupling of neurotransmitter release to action potentials (Bruckner, 2016).

The specific neurotransmitter release properties of an active zone are determined by two key parameters acting in concert: (1) the number of synaptic vesicles docked at the membrane and molecularly primed for Ca2+-triggered release, termed the readily releasable pool, and (2) the release probability of these vesicles. Vesicle release probability is established by multiple parameters, including Ca2+ channel levels, localization and function at active zones, the spatial coupling of Ca2+ channels and release-ready vesicles, and the intrinsic Ca2+ sensitivity of individual vesicles. The observation that the presynaptic parameters of synaptic strength vary significantly even between the synapses of an individual neuron indicates that neurotransmitter release properties are determined locally at active zones and raises the question of how this complex regulation is achieved. Genetic studies in Drosophila, Caenorhabditis elegans, and mice are revealing a key role for the active zone cytomatrix in determining the functional parameters underlying synaptic strength (Ackermann, 2015; Michel, 2015). A mechanistic understanding of how the active zone cytomatrix achieves local control of synaptic release properties will yield fundamental insights into neural circuit function (Bruckner, 2016).

Fife, a Piccolo-RIM-related protein is required for proper neurotransmitter release and motor behavior (Bruckner, 2012). This study demonstrates that Fife localizes to the active zone cytomatrix, where it interacts with RIM to promote neurotransmitter release. The active zone cytomatrix is diminished and molecularly disorganized at Fife mutant synapses, and Fife is critical for vesicle docking at the active zone membrane. Not only are the number of release-ready vesicles reduced in the absence of Fife, but their probability of release is also significantly impaired because of disrupted coupling to calcium channels. These results suggest that Fife promotes high-probability neurotransmitter release by organizing the active zone cytomatrix to create vesicle release sites in nanometer proximity to clustered Ca2+ channels. Finally, it was found that in addition to its role in determining baseline synaptic strength, Fife plays an essential role in presynaptic homeostatic plasticity. Together, these findings provide mechanistic insight into how synaptic strength is established and modified to tune communication in neural circuits (Bruckner, 2016).

This study demonstrates that Fife plays a key role in organizing presynaptic terminals to determine synaptic release properties. Fife functions with RIM at the active zone cytomatrix to promote neurotransmitter release. Functional and ultrastructural imaging studies demonstrate that Fife regulates the docking of release-ready synaptic vesicles and, through nanodomain coupling to Ca2+ channels, their high probability of release. It was further found that Fife is required for the homeostatic increase in neurotransmitter release that maintains circuit function when postsynaptic receptors are disrupted. These findings uncover Fife's role as a local determinant of synaptic strength and add to understanding of how precise communication in neural circuits is established and modulated (Bruckner, 2016).

The finding that Fife interacts with RIM provides insight into how Fife functions within the network of cytomatrix proteins. RIM is a central active zone protein that was recently shown to facilitate vesicle priming at mammalian synapses by relieving autoinhibition of the priming factor Munc13 (Deng, 2011). Like Fife, Drosophila RIM promotes Ca2+ channel accumulation at active zones and exhibits EGTA-sensitive neurotransmitter release at the Drosophila NMJ. This suggests that Fife and RIM may promote high-probability neurotransmitter release by acting together to dock and prime synaptic vesicles in close proximity to Ca2+ channels clustered at the cytomatrix. The findings are consistent with previous work in pancreatic β cells, where it was found that Piccolo and RIM2α form a complex that promotes insulin secretion through an unknown mechanism. To date, functional studies at mammalian synapses have focused on investigating interactions between Piccolo and Bassoon, which bind through their common coiled-coil regions. Thus, it will be of interest to investigate the functional relationship between Piccolo and RIM in promoting neurotransmitter release in mouse models. Piccolo also binds CAST1 through coiled-coil domain interactions. Although the Piccolo coiled-coil region is not present in Fife, future studies to determine whether this interaction is preserved through distinct interacting domains will be important, as Fife and Drosophila CAST-related Bruchpilot carry out overlapping functions. Similarly, although neither Fife nor Piccolo contains the conserved SH3-binding domain that mediates the interaction between RIM and RIM-binding protein, the overlap between Fife and RIM-binding protein phenotypes raises the possibility of functional interactions that will also be important to investigate in future experiments (Bruckner, 2016).

Significant alterations to active zone cytomatrix size and structure were observed in Fife mutants, whereas none have been detected in RIM mutants, indicating that Fife carries out this function independently of RIM. Previous ultrastructural analysis in aldehyde-fixed samples revealed occasional free-floating electron-dense structures that resemble active zone cytomatrix material and cluster synaptic vesicles (Bruckner, 2012). These unanchored electron-dense structures have also been observed at low frequency in Drosophila RIM-binding protein mutants, which, like Fife mutants, exhibit smaller active zone cytomatrices, and at higher frequency in rodent ribbon synapses lacking Bassoon. These structures were not visible in HPF/FS-prepared electron microscopy samples, likely because protein components of the synapse are not cross-linked upon fixation. That these structures are not observed in control synapses but have been found in multiple active zone cytomatrix mutants argues that the extensive cross-linking of proteins in chemically fixed preparations may enable the visualization of biologically relevant complexes missed with cryofixation. This supports the idea that the two fixation methods may offer different advantages for ultrastructural studies of synapses. In any case, the active zone cytomatrix is significantly reduced in size at Fife active zones in both HPF/FS- and aldehyde-fixed electron micrographs (Bruckner, 2016).

Although diminished or absent cytomatrices have been observed in electron micrographs of RIM-binding protein and Bruchpilot mutants, this phenotype has not been observed in electron micrographs of other active zone cytomatrix mutants, suggesting it represents more than the loss of a single component protein. Rather, the reduced complexity visible in electron micrographs likely reflects a broader underlying molecular disorganization. Further support for this model comes from superresolution imaging, which reveals molecular disorganization at Fife active zones as indicated by the loss of the characteristic ring-shaped localization pattern of Bruchpilot's C terminus. Similar disorganization of Bruchpilot was observed at active zones lacking RIM-binding protein. Superresolution imaging was used to investigate the localization of active zone proteins Cacophony and RIM-binding protein and, although the levels of Cacophony are reduced at Fife active zones, no apparent differences were observed in the patterns of these proteins. Cacophony and RIM-binding protein both localize in smaller puncta than Bruchpilot, so future studies with higher resolution imaging modalities such as stimulated emission depletion microscopy may reveal more subtle abnormalities. Correlations between active zone molecular composition and release probability have been observed at diverse synapses. At the Drosophila NMJ, functional imaging with genetically encoded Ca2+ indicators has demonstrated that active zones display a wide range of release probabilities. Active zones with high release probability contain higher levels of Bruchpilot, which may in turn correlate with higher Ca2+ channel levels. At mouse hippocampal synapses, Bassoon and RIM levels directly correlate with neurotransmitter release probability. Consistently, synaptic probability of release is significantly decreased in Fife mutants (Bruckner, 2016)

Through a combination of morphological and functional studies, this study found that Fife acts to promote the active zone docking of synaptic vesicles and regulates their probability of release. Because the number of readily releasable vesicles appears to scale with active zone cytomatrix size and molecular composition at diverse synapses, a conserved function of the active zone cytomatrix may be to establish release sites for synaptic vesicles. Consistent with this view, the number of release-ready vesicles is also reduced in Drosophila RIM-binding protein-null mutants and isoform-specific bruchpilotΔ170 and bruchpilotΔ190 mutants, which share similar active zone structural abnormalities with Fife. By combining rapid preservation of intact Drosophila larvae by HPF/FS fixation, electron tomography, and extensive segmentation of active zone structures, this study obtained a detailed view of the 3D organization of active zones in near-native state that allowed further dissection of Fife's role in determining the size of the readily releasable vesicle pool. Membrane-docked vesicles are significantly decreased in Fife mutants, whereas more distant vesicles attached to the membrane by long tethers appear unaffected. Correlating physiological and morphological parameters of neurotransmission is an ongoing challenge in the field. It has been proposed that docking and priming are not separable events in the establishment of the readily releasable vesicle pool, but rather the morphological and physiological manifestations of a single process. Although approximately one third of docked vesicles in these preparations lack obvious short connections to the membrane, which are thought to represent priming factors, the possibility cannot be excluded that these filaments are present but obscured, perhaps because the vesicles are more tightly linked to the membrane. As this proportion is unchanged in Fife mutants, it is concluded that Fife acts to promote vesicle docking and may simultaneously facilitate molecular priming-possibly through its interactions with RIM (Bruckner, 2016).

The data indicate that neurotransmitter release at Fife synapses is highly sensitive to EGTA, a slow Ca2+ chelator that has been used to investigate the coupling of Ca2+ influx at voltage-gated Ca2+ channels and Ca2+ sensors on synaptic vesicles. At synapses with high release probability, including inhibitory synapses in the mammalian hippocampus and cerebellum, excitatory synapses of the mature Calyx of held, and the Drosophila NMJ, molecularly primed synaptic vesicles and Ca2+ channel clusters are thought to be positioned within ~100 nm of one another to ensure the tight coupling of Ca2+ influx and Ca2+ sensors that explains observed release properties. The EGTA sensitivity of release at Fife, but not wild-type, NMJs indicates that Fife likely regulates the probability that a docked vesicle is released by positionally coupling release-ready vesicles to Ca2+ channels clustered beneath the active zone cytomatrix. The trend toward fewer docked vesicles associated with the active zone cytomatrix in tomograms of Fife synapses provides morphological support for this model. Building on detailed tomographic studies of the Drosophila NMJ to visualize how Ca2+ channels and vesicles are spatially organized at active zones in different genetic backgrounds will be an important step in advancing understanding of the geometry of release probability and how it is established (Bruckner, 2016).

Finally, this study found that Fife is required for presynaptic homeostasis. In response to decreases in glutamate receptor levels or function, Drosophila motoneurons rapidly increase synaptic vesicle release to maintain postsynaptic excitation. This homeostatic increase in presynaptic neurotransmission is accompanied by an increase in the number of dense projections per active zone and Bruchpilot levels. Cytomatrix proteins RIM, RIM-binding protein, and now Fife have all been shown to function in presynaptic homeostasis, indicating a critical role for the active zone cytomatrix as a substrate for synaptic plasticity. These studies provide insight into the molecular mechanisms through which the active zone cytomatrix determines neurotransmitter release parameters to modulate how information flows in neural circuits (Bruckner, 2016).

Neuronal communication depends on the precisely orchestrated release of neurotransmitter at specialized sites called active zones (AZs). A small number of scaffolding and cytoskeletal proteins comprising the cytomatrix of the active zone (CAZ) are thought to organize the architecture and functional properties of AZs. The majority of CAZ proteins are evolutionarily conserved, underscoring the fundamental similarities in neurotransmission at all synapses. However, core CAZ proteins Piccolo and Bassoon have long been believed exclusive to vertebrates, raising intriguing questions about the conservation of the molecular mechanisms that regulate presynaptic properties. This study presents the identification of a piccolo-rim-related gene in invertebrates, together with molecular phylogenetic analyses that indicate the encoded proteins may represent Piccolo orthologs. In accordance, the Drosophila homolog, Fife, was found to be neuronal and localizes to presynaptic AZs. To investigate the in vivo function of Fife, a deletion of the fife locus was generated. Evoked neurotransmitter release is substantially decreased in fife mutants and loss of fife results in motor deficits. Through morphological analysis of fife synapses, underlying AZ abnormalities were identified including pervasive presynaptic membrane detachments and reduced synaptic vesicle clustering. These data demonstrate the conservation of a Piccolo-related protein in invertebrates and identify critical roles for Fife in regulating AZ structure and function. These findings suggest the CAZ is more conserved than previously thought, and open the door to a more complete understanding of how CAZ proteins regulate presynaptic structure and function through genetic studies in simpler model systems (Bruckner, 2012).

The CAZ is a conserved molecular machine responsible for coordinating the structure and function of presynaptic terminals. With the notable exceptions of Piccolo and Bassoon, representatives of all major vertebrate CAZ protein families have been found in invertebrates. This study presents the identification of a previously overlooked invertebrate Piccolo-RIM homolog. A requirement is uncovered for the Drosophila homolog, Fife, in locomotor behavior and neurotransmitter release, and a role is defined for Fife in AZ organization and synaptic vesicle clustering at the NMJ (Bruckner, 2012).

Is Fife a Piccolo ortholog or a second RIM ortholog? Phylogenetic analyses support the former model. However, Fife, which exhibits significant sequence homology to both Piccolo and RIM proteins, is structurally more similar to RIMs. Because molecular phylogenetic approaches rely on conserved sequences, the nonconserved CC regions are not considered in these analyses. Determining orthology based on function is likely to prove similarly complicated in this case because CAZ proteins share many physical interactions and often function redundantly in carrying out core CAZ functions. Future studies of protein-protein interactions may prove informative. Previous work in yeast has demonstrated that combining sequence-level analysis with protein-protein interaction data improves the ability to identify functional orthologs. While RIMs and Piccolo have many binding partners in common, they also have unique associations. Determining which Piccolo/RIM associations are conserved in Fife will be an important step toward understanding the conservation of CAZ function in general and Fife function specifically (Bruckner, 2012).

As noted, molecular and computational searches in Drosophila have not identified sequences that encode the large CC domain-containing region of Piccolo, either at the fife locus or elsewhere in the genome, suggesting that this sequence is specific to vertebrates or was lost in the insect lineage. Interestingly, while insect Fife proteins may have lost their CC domains, the opposite is true of the Drosophila CAST homolog Bruchpilot, which colocalizes with Fife at AZs and has a much larger CC-containing region than its vertebrate counterparts. These observations raise the intriguing possibility that the expansion of Bruchpilot's CC regions may have reduced selective pressure to maintain these regions in Fife, and that evolutionary conservation may be found at the level of the intertwined complex of CAZ proteins as well as at the level of individual proteins (Bruckner, 2012).

The CC domains of Piccolo mediate physical interactions with CAZ proteins CAST and UNC-13. However, it is interesting to note that both of these physical interactions are maintained in RIM proteins despite the lack of CC domains as RIM binds CAST through its PDZ domain and UNC-13 through its ZF domain. Future studies will be important for determining if physical interactions mediated by the CC domains of vertebrate Piccolo are performed by other domains in Fife or by other CAZ proteins such as Bruchpilot (Bruckner, 2012).

The CC domain-containing region of Piccolo gives the protein its large size. If Piccolo's 5100 aa were arranged in an extended α-helical conformation it could in principle span 750 nm. It has long been speculated that this potential, also present in Bassoon, has important implications for Piccolo-Bassoon function. However, in a recent immunoelectron microscopy study, the localization of 11 Piccolo epitopes spanning the length of the protein were determined to understand its spatial organization at AZs. Piccolo adopts a compact conformation with its N terminus localizing near-dense projections ~75 nm from the plasma membrane and its C terminus localizing within 30-40 nm of the plasma membrane in close proximity to binding partners RIM and UNC-13. This finding is supported by a study employing stochastic optical reconstruction microscopy, and argues that the functions of Piccolo are not dependent upon its large size but rather, like RIMs, on its many interactions with other AZ proteins-a role that is likely maintained in Fife (Bruckner, 2012).

To investigate Fife function at Drosophila synapses, deletion alleles were generated. This study focused on fifeex1027, a deletion mutant that behaves as a genetic null in that its phenotype over a chromosomal deficiency mirrors its homozygous phenotype in morphological, functional, and behavioral assays. Loss of Fife results in a 50% reduction in locomotion and 60% decrease in evoked neurotransmitter release. To uncover the underlying cause of these deficits, a morphological analysis was performed of fife synapses that revealed crucial roles for Fife in the regulation of AZ structure. Absolute number and gross morphology of synapses is largely normal in fife mutants. This is consistent with functional studies of Piccolo and RIM proteins demonstrating normal formation of synapses and the accumulation of wild-type levels of synaptic proteins in their absence. However, fife AZs often exhibit separations of presynaptic and postsynaptic membranes as revealed by ruffling of the presynaptic membrane in electron micrographs. As this defect was observed along much of the length of affected AZs, it likely disrupts presynaptic release sites and thus contributes to the deficit in exocytosis observed at fife NMJs. This phenotype has previously been linked to decreased cell adhesion, raising the possibility that Fife might regulate the levels or localization of trans-synaptic cell adhesion molecules at synapses. Less frequently floating electron densities were observed, a phenotype highly reminiscent of the floating ribbons observed at bassoon mutant ribbon synapses in the vertebrate retina. At ribbon synapses, electron dense ribbon-shaped structures extend from the AZ membrane into the presynaptic cytoplasm and, like T-bars, cluster synaptic vesicles. In Drosophila, floating electron densities have also been observed at low frequency in the absence of Rim-Binding Protein, a CAZ-associated protein with an essential role in neurotransmitter release. It will be important to determine whether Fife functions with Rim-Binding Protein and/or other CAZ proteins to control the structural integrity of AZs (Bruckner, 2012).

A role was identified for Fife in clustering synaptic vesicles. At fife synapses, 20% fewer synaptic vesicles reside within 200 nm of the AZ membrane than at control synapses. In their study of piccolo knock-out mice, a similar deficit in synaptic vesicle clustering at cortical synapses lacking both Piccolo and Bassoon, as did a recent study of Bassoon function at mouse hair cell ribbon synapses. It will be of interest to determine if this deficit at fife AZs includes a decrease in the number of docked (membrane-bound) vesicles as has been observed in rim and bassoon mutants and in piccolo, bassoon double knockdown neurons. This study quantified docked vesicles in electron microscopic samples, which were prepared by conventional aldehyde fixation methods, and no significant difference was observed between wild-type and fife mutants. However, recent studies in mice and C. elegans have made it clear that aldehyde fixation masks vesicle-docking defects. Thus, future studies employing high-pressure freeze fixation methods will be required to determine whether Fife functions in the docking of vesicles for rapid release (Bruckner, 2012).

The number and nature of vertebrate Piccolo/RIM-binding partners hint at additional roles for Fife at AZs. However, it is currently unknown if many of these vertebrate interactions are biologically relevant. With the identification of a Piccolo-related protein in invertebrates and the generation of fife alleles in Drosophila, this question can now be readily addressed through double mutant analysis. Studies of CAZ components in Drosophila and C. elegans have been critical to understanding of AZ organization and function. This study reveals greater conservation of CAZ proteins than previously believed and opens the door to a more complete understanding of CAZ function through in vivo studies of this fundamental molecular machine and its core components in an amenable genetic model system (Bruckner, 2012).